REG1A Antibody

What is REG1A and where is it primarily expressed in human tissues?

REG1A, also known as lithostathine, is a secreted protein with a C-type lectin domain. It is primarily expressed in the pancreas, specifically in exocrine cells. Due to variable glycosylation, pancreatic REG1A exists as multiple species of 16-18 kDa . The protein promotes the maintenance and growth of pancreatic islet beta-cells and intestinal villi. REG1A is also present in a distinct type of acinar-like cell clusters touching Langerhans islets (ATLANTIS) that secrete REG1A-containing vesicles to neighboring islet cells in non-diabetic human pancreas . Beyond the pancreas, REG1A expression has been reported in various tissues under pathological conditions, including gastrointestinal tract, minor salivary glands, and certain cancer types .

How can I distinguish between REG1A and REG1B in experimental systems?

REG1A and REG1B share 87% sequence homology, making their differentiation challenging . When using antibodies, careful selection is essential as some antibodies cross-react with both proteins. For instance, the antibody MAB49371 shows 100% cross-reactivity with recombinant human REG1B in Western blot . For experiments requiring specific detection of REG1A alone:

• Use validated antibodies with confirmed specificity

• Employ recombinant REG1A and REG1B as positive controls to assess cross-reactivity

• Consider advanced techniques such as:

  • Mass spectrometry for protein identification

  • Custom antibody development against unique epitopes

  • RNA-based methods (RT-PCR, RNA-seq) to distinguish at the transcript level

What are the recommended applications for REG1A antibody detection?

Based on validated applications from multiple sources:

For optimal results, each laboratory should determine the appropriate dilutions for their specific applications .

What are the optimal tissue preparation methods for REG1A immunohistochemistry?

For successful REG1A detection in tissues, follow these validated protocols:

• Fixation: Immersion-fixed paraffin-embedded sections yield reliable results

• Epitope retrieval: Heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic is critical before antibody incubation

• Antibody incubation:

  • For MAB49371: Use at 15 μg/mL overnight at 4°C

  • For AF4937: Use at 3 μg/mL overnight at 4°C

• Detection systems:

  • With rat monoclonal antibodies: Anti-Rat HRP-DAB Cell & Tissue Staining Kit

  • With sheep polyclonal antibodies: Anti-Sheep HRP-DAB Cell & Tissue Staining Kit

• Counterstaining: Hematoxylin provides good nuclear contrast

This approach reveals specific staining in exocrine cells of the pancreas, with AF4937 specifically localizing to the cytoplasm of these cells .

How should REG1A antibodies be stored and handled to maintain activity?

To preserve antibody functionality:

• Short-term storage (immediate use, up to two weeks): 4°C is acceptable

• Long-term storage: Divide into small aliquots (≥20 μL) and store at -20°C or -80°C

• Avoid freeze-thaw cycles, which can degrade antibody quality

• For concentrate products, consider adding an equal volume of glycerol as a cryoprotectant before freezing

• Shelf-life:

  • As supplied: 12 months from receipt date at -20 to -70°C

  • After reconstitution: 1 month at 2-8°C under sterile conditions

  • After reconstitution with cryoprotection: 6 months at -20 to -70°C under sterile conditions

These storage recommendations help maintain antibody specificity and sensitivity for experimental applications.

How can REG1A antibodies be used to study diabetes pathogenesis and progression?

REG1A has significant implications in diabetes research, with distinct expression patterns across diabetes types:

• Serum biomarker analysis:

  • Increased circulating serum levels of REG1A are observed in type 1 diabetes (T1D), maturity onset diabetes of the young (MODY), and type 2 diabetes (T2D)

  • Use sandwich ELISA with a combination of monoclonal (e.g., MAB49371) and polyclonal (e.g., AF4937) antibodies for serum quantification

• Temporal analysis:

  • REG1A levels correlate with disease duration in T2D and MODY, but not in T1D

  • Longitudinal measurements can provide insights into disease progression

• Autoimmunity studies:

  • T1D patients with increased REG1A exhibit significantly higher levels of corresponding autoantibodies than non-diabetic and T2D subjects

  • Combined analysis of REG1A levels and autoantibodies may aid in understanding autoimmune processes

• Pancreatic islet-acinar interactions:

  • Investigate ATLANTIS cell clusters, which secrete REG1A-containing vesicles to neighboring islet cells

  • Use immunohistochemistry to study REG1A overexpression in ATLANTIS under diabetic conditions

• Therapeutic response monitoring:

  • REG1A upregulation after gastric bypass surgery may contribute to T2D remission

  • Serial measurements can track regenerative responses to interventions

What are the applications of REG1A antibodies in cancer research?

REG1A expression has been reported in multiple cancer types with varying prognostic implications:

Research methodologies include:

• Immunohistochemical profiling of tumor tissues using validated antibodies (MAB49371, AF4937)

• Western blot analysis of tumor lysates to quantify REG1A protein levels

• Correlation of REG1A expression with clinical outcomes and treatment responses

• Functional studies exploring the role of REG1A in tumor growth, invasion, and apoptosis

The seemingly contradictory findings in gastric cancer, where both tumor promotion and apoptosis have been associated with REG1A, highlight the need for context-specific investigation .

How can I design experiments to investigate REG1A's role in tissue regeneration?

REG1A's name (Regenerating Islet-Derived Protein) reflects its regenerative potential in multiple tissues:

• Pancreatic regeneration models:

  • Use antibodies to track REG1A expression during pancreatic recovery after injury

  • Correlate REG1A levels with beta-cell proliferation markers

  • Apply immunohistochemistry to locate REG1A in regenerating tissues

• Gastrointestinal healing:

  • Investigate REG1A's protective role against NSAID-induced bowel damage

  • Study REG1A expression during recovery from parasitic infections like amoebic colitis

  • Monitor REG1A levels during mucosal healing in inflammatory bowel conditions

• Wound healing studies:

  • Examine REG1A's role in keratinocyte proliferation and differentiation after skin injury

  • Use antibodies to track REG1A expression in healing tissues

  • Correlate with other wound healing markers

• Bone regeneration:

  • Investigate REG1A expression in periosteum during bone fracture healing

  • Study IL-6-induced REG expression in periosteum-derived mesenchymal stem cells

For experimental approaches, consider:

• Loss-of-function studies (siRNA, CRISPR/Cas9)

• Gain-of-function experiments (recombinant protein administration, overexpression)

• Cell-specific conditional knockouts to elucidate tissue-specific functions

• Combined immunofluorescence to co-localize REG1A with proliferation markers

Why might I observe multiple bands in Western blot when using REG1A antibodies?

Multiple bands in REG1A Western blots can occur for several reasons:

• Post-translational modifications:

  • REG1A undergoes variable glycosylation, resulting in multiple species of 16-18 kDa

  • Pancreatic REG1A exists as multiple species due to these modifications

• Cross-reactivity:

  • Some antibodies (e.g., MAB49371) show cross-reactivity with REG1B (87% homologous to REG1A)

  • Check the antibody specifications for known cross-reactivity

• Protein degradation:

  • Proteolytic fragments can appear as lower molecular weight bands

  • Use fresh samples and protease inhibitors during extraction

• Experimental conditions:

  • Reducing vs. non-reducing conditions can affect band patterns

  • AF4937 detects specific bands at approximately 20-22 kDa under reducing conditions

To address these issues:

• Include positive controls with recombinant REG1A protein

• Perform peptide competition assays to verify specificity

• Test different antibodies (monoclonal vs. polyclonal)

• Consider deglycosylation experiments to eliminate glycoform heterogeneity

What are the best validation approaches for REG1A antibody specificity?

Rigorous validation is essential for confident interpretation of REG1A antibody results:

• Multiple antibody approach:

  • Use antibodies from different sources targeting different epitopes

  • Compare monoclonal (MAB49371, MMC-Reg1a-4F1) and polyclonal (AF4937) antibodies

• Recombinant protein controls:

  • Test antibody against purified recombinant REG1A

  • Include REG1B to assess cross-reactivity (especially important since MAB49371 shows 100% cross-reactivity with recombinant human REG1B)

• Tissue controls:

  • Human pancreas serves as a positive control tissue

  • Include REG1A-negative tissues as negative controls

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide or recombinant protein

  • Should abolish specific staining in Western blot or IHC

• Genetic models:

  • Use tissues from REG1A knockout models as negative controls

  • Overexpression systems can serve as positive controls

• Orthogonal techniques:

  • Validate findings using mRNA detection methods (RT-PCR, RNA-seq)

  • Mass spectrometry for protein identification

  • Multiple antibody-based techniques (Western, IHC, ELISA)

These validation strategies ensure reliable results and help distinguish between REG1A and closely related family members.

How can REG1A antibodies be utilized in autoimmune disease research?

REG1A has implications in several autoimmune conditions:

• Type 1 diabetes (T1D):

  • REG1A is an antigenic target in autoimmune diabetes

  • T1D patients with increased REG1A show significantly higher levels of corresponding autoantibodies

  • Research applications include:

    • Monitoring REG1A antibodies as potential biomarkers of beta-cell stress

    • Investigating the relationship between REG1A expression and autoimmune attack

    • Studying REG1A's potential role in beta-cell regeneration efforts

• Sjögren's Syndrome (SS):

  • REG1A is upregulated in minor salivary glands of SS patients

  • Believed to play a role in regeneration of ductal cells

  • Research approaches include:

    • Immunohistochemical analysis of salivary gland biopsies

    • Correlation of REG1A expression with disease severity and duration

    • Investigation of regenerative responses in damaged salivary tissue

• Celiac disease:

  • Serum REG1A levels are over two-fold higher than normal

  • Levels decrease with adoption of a gluten-free diet

  • Applications include:

    • Monitoring disease activity and response to dietary intervention

    • Studying intestinal regeneration processes

    • Evaluating REG1A as a non-invasive biomarker of intestinal damage

• Methodological considerations:

  • Combine tissue immunohistochemistry with serum biomarker analysis

  • Follow longitudinal changes in REG1A levels during disease progression and treatment

  • Correlate REG1A expression with inflammatory markers and clinical outcomes

What methodological approaches are recommended for studying REG1A in multiple tissue contexts?

Given REG1A's diverse tissue expression and functions, tailored approaches are needed:

• Pancreatic tissue analysis:

  • IHC with MAB49371 (15 μg/mL) reveals staining in exocrine cells

  • IHC with AF4937 (3 μg/mL) shows cytoplasmic staining in exocrine cells

  • Heat-induced epitope retrieval is essential for optimal staining

• Serum/plasma quantification:

  • Combination of monoclonal (MAB49371) and polyclonal (AF4937) antibodies in sandwich ELISA provides sensitive detection

  • Both antibodies have been validated for human serum and plasma samples

• Gastrointestinal tissues:

  • Study REG1A expression in relation to intestinal villus growth and maintenance

  • Investigate protective effects against NSAID damage and parasitic infections

• Cancer tissues:

  • Compare REG1A expression in tumor vs. adjacent normal tissue

  • Correlate with clinicopathological features and prognosis

  • Consider context-specific functions (may promote or inhibit tumor growth depending on cancer type)

• Wound healing and regeneration:

  • Track temporal expression during healing processes

  • Correlate with regenerative markers and functional recovery

  • Consider IL-6-induced REG expression pathways

• Comparative analysis across tissues:

  • Standardize detection methods for cross-tissue comparison

  • Consider tissue-specific post-translational modifications

  • Explore tissue-specific signaling partners and downstream effects

These methodological considerations enable robust investigation of REG1A's diverse biological roles across multiple physiological and pathological contexts.